Miniprep/Kit-free high-throughput protocol

Contents

Background

This protocol is adapted from "Molecular Cloning: A Laboratory Manual", Second Edition, Sambrook, Fritsch, and Maniatis. It is a quick, inexpensive way to purify large numbers of plasmids (I used to routinely do 80 at a time.--Kathleen) and yields DNA that is clean enough for sequencing or for use as a PCR template.

Protocol

Transfer 1.5 mL of an overnight culture containing your plasmid to an eppendorf tube and spin at 5000 rpm for 5 min in a tabletop centrifuge to pellet the cells.

Remove pellet from each tube with a toothpick. The cellular debris should stick well to the toothpick. Try to insert and remove the toothpick from the center of the tube so you don't get any cellular debris on the sides of the tube.

Add 300 μL ice cold isopropanol to precipitate the DNA (or 300μL of 2:1 isopropanol:ammonium acetate, mixed just before you use it. See this discussion of precipitating nucleic acids.)

Dry pellet (air dry at room temperature or 37 ˚C or dry in a speedvac).

Rehydrate in 50 μL TE.

Buffers

STET

8% sucrose
50 mM Tris-HCl, pH 8
0.5% Triton X-100
50 mM EDTA

TE

10 mM Tris-HCl, pH 8
1 mM EDTA

BioCoder version

Following is the Miniprep/Kit-free high throughput protocol in BioCoder, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioCoder (see Source code). More information about BioCoder can be found on my home page. Feel free to mail me your comments/ suggestions.Vaishnavi